Publikationsdatenbank

Analysis of hydrostatic pressure effects on the barrier properties of mammary epithelial cells (2018)

Art

Hochschulschrift

Autor

Mießler, Katharina Sophie (WE 2)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2018 — VIII, 72 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/23618

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

During lactation, the cycle of milk synthesis and removal by suckling or milking causes high hydrostatic pressure differences in the mammary gland. To prevent an uncontrolled exchange of milk, blood and intestinal fluid, the mammary gland tissue maintains the bloodmilk barrier, which on paracellular level is controlled by the tight junction. In previous studies, Markov et al. (2012) found an upregulation of tightening and a downregulation of permeability-mediating tight junction proteins after cessation of milk removal in mice. The present thesis investigated if and how hydrostatic pressure can mechanically affect the mammary epithelial barrier function. With respect to in vivo-approaches as performed by Markov et al. (2012), the present thesis further aimed to analyze pressure induced effects isolated from the influence of milk compounds, in vitro. To accomplish this, a method was successfully established to analyze barrier properties during dynamical changes of hydrostatic pressure. For functional analyses of epithelial barrier properties, the Ussing chamber has been proved to be a powerful experimental technique. Therefore, an Ussing chamber was modified with an additional tube-system to apply hydrostatic pressure to the apical, basal or both sides of an epithelial monolayer, in a range of 1 to 10 kPa. The chamber was provided with a manometer and a syringe to dynamically change the pressure during experiments. Employing the maximal pressure level, stable experimental conditions regarding pH and buffer distribution were confirmed. In the first part of this thesis, effects of negative pressure on the mammary epithelial barrier function were investigated, simulated by increasing basal pressure to monolayers of the mammary epithelial cell line HC11. A combination of different cultivation times (7 and 14 days) and incubation steps (5 and 30 minutes) was employed to further investigate maturing processes and time-dependent adaptation. The basal pressure increase resulted in an immediate and strong decrease of the transepithelial electrical resistance (RT) in all approaches. In parallel, a successive increase of the short circuit current (ISC) was observed during five steps of +1 kPa basal hydrostatic pressure application. Furthermore, a partial recovery was shown in a following equalization step, and the epithelial monolayer integrity was confirmed via immunohistochemical staining of the tight junction. Although the molecular origin remains to be clarified, the obtained data strongly suggest that negative pressure induces protective mechanisms in the mammary epithelial barrier function. To further elucidate possible adaptive mechanisms of the epithelial barrier during milk stasis within lactation, the second part of the thesis focused on bilateral pressure incubation of HC11 monolayers. 14 day-cultivated HC11 cells were incubated with 10 kPa bilateral hydrostatic pressure for 4 hours and compared with hormone induced HC11 cells in a parallel approach, differentiated by treatment with prolactin and dexamethasone. Whereas no significant changes of RT were observed, a decrease of ISC was shown in the differentiated cells, which could partly be inhibited by a barium chloride-induced blockage of inward rectifier potassium channels. Moreover, molecular analyses revealed an upregulation of the scaffolding protein ZO-1 in differentiated cells, which was downregulated after an inhibition with barium chloride. Taken together, it can be assumed by the obtained data that a pressure induced chloride transport possibly was mediated by mechanosensitive transduction, while changes of ZO-1 expression might indicate an interaction of transport and barrier mechanisms. In conclusion, dynamical pressure studies on the mammary epithelial barrier function became assessable by the modified Ussing chamber, which was established as part of this thesis. Moreover, the method may be employed on other cell monolayers and epithelial tissues in future experiments, representing a new tool to study pressure effects, in vitro. From the results acquired in both parts of this thesis, it can be conducted that mammary epithelium reacts to hydrostatic pressure with functional and adaptive processes in barrier function, involving tight junction protein composition and transcellular transport mechanisms. These findings elucidate the physiological processes in mammary barrier function and underline the importance to maintain the blood-milk barrier during pressure conditions induced by suckling, milking and times of milk accumulation in the mammary gland.